Cholesterol oxidase senses subtle changes in lipid bilayer structure
Cholesterol oxidase senses subtle changes in lipid bilayer structure
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DOI：
10.1021/bi035697q
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发表时间：
2004-01-27
期刊：
影响因子：
2.9
通讯作者：
Sampson, NS
中科院分区：
文献类型：
--
作者：
Ahn, KW;Sampson, NS
我们研究了胆固醇氧化酶的催化活性和膜亲和性的依赖模型膜双层脂质结构。胆固醇氧化酶的结合亲和力的100 nm的单层囊泡组成的混合物的DOPC或DPPC和胆固醇是不敏感的胆固醇摩尔分数，如果膜的相是在流体状态。当膜处于固体有序状态时，胆固醇氧化酶的结合亲和力增加约10倍。不同脂质混合物的二阶速率常数（k（cat）*/K-m*）显示出对l（d）相中的胆固醇的2倍底物特异性，其胆固醇化学活性高于l（o）相中的胆固醇。此外，该酶在l（o）相中对胆固醇的特异性是s（o）相中的2倍。同样，高胆固醇化学活性Id相的底物特异性是低化学活性Id相的2倍。低胆固醇化学活性的l（d）相和l（o）相的特异性相同。这些数据表明，双层中的脂质胆固醇结构越有序，催化速率越低。然而，在所有研究的条件下，酶从未被底物饱和。酶活性直接反映了胆固醇可以移出膜的能力，胆固醇转移能力的变化是否是由于相变或更局部的包装变化。我们的结论是胆固醇氧化酶的活性是直接和敏感地依赖于其底物结合的膜的物理性质。
We investigated the dependence of cholesterol oxidase catalytic activity and membrane affinity on lipid structure in model membrane bilayers. The binding affinities of cholesterol oxidase to 100-nm unilamellar vesicles composed of mixtures of DOPC or DPPC and cholesterol are not sensitive to cholesterol mole fraction if the phase of the membrane is in a fluid state. When the membrane is in a solid-ordered state, the binding affinity of cholesterol oxidase increases approximately 10-fold. The second-order rate constants (k(cat)*/K-m*) for different lipid mixtures show a 2-fold substrate specificity for cholesterol in the l(d) phase of high cholesterol chemical activity over cholesterol in the l(o) phase. Moreover, the enzyme is 2-fold more specific for cholesterol in the l(o) phase than in the s(o) phase. Likewise, there is 2-fold substrate specificity for the high cholesterol chemical activity ld phase over the low chemical activity Id phase. The specificities for the l(d) phase of low cholesterol chemical activity and the l(o) phase are the same. These data indicate that the more ordered the lipid cholesterol structure in the bilayer, the lower the catalytic rate. However, under all of the conditions investigated, the enzyme is never saturated with substrate. The enzymatic activity directly reflects the facility with which cholesterol can move out of the membrane, whether changes in cholesterol transfer facility are due to phase changes or more localized changes in packing. We conclude that the activity of cholesterol oxidase is directly and sensitively dependent on the physical properties of the membrane in which its substrate is bound.